Targeted particles and methods of using the same

ABSTRACT

Drug delivery compositions and methods of delivering compounds to are disclosed. Vaccines and methods of immunizing individuals are disclosed. Compositions for drug delivery including gene therapy and methods of treating individuals using such compositions are disclosed.

FIELD OF THE INVENTION

The present invention relates to drug delivery compositions, to methodsof delivering compounds to specific cell types, to vaccines, to methodsof immunizing individuals, to compositions for drug delivery includinggene therapy and to methods of treating individuals using suchcompositions.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,714,316, which is incorporated herein by reference,describes the design and production of viral particles which displayheterologous protein sequences on the viral particle envelope.

U.S. Pat. Nos. 4,873,089, 5,227,470 and 5,258,499, which areincorporated herein by reference, describe methods of preparingliposomes that contain proteins displayed on their surfaces in order totarget the liposomes to a cell with a cellular protein on its surfacethat specifically binds to the protein on the surface of the liposome.

U.S. Pat. Nos. 5,837,533, 5,459,127 and Behr, J. P., et al. (1989) Proc.Natl. Acad. Sci. USA 86:6982-6986, which are each incorporated herein byreference, describe the design and production of receptor targetedcationic amphiphile/DNA complexes in which positively charged lipophiliccompounds are provided with receptor ligands. The cationic amphiphiliccompounds contain receptor ligand moieties which are displayed on thesurface of complexes formed when the cationic amphiphile is mixed withDNA. Such teachings may also be applied to cationic lipid/DNA complexessuch as those described in U.S. Pat. Nos. 5,955,365, 5,948,767,5,945,400, 5,939,401, 5,935,936, 5,932,241, 5,925,628, 5,916,803,5,910,488, 5,908,635, 5,891,468, 5,885,613, 5,830,430, 5,827,703,5,783,565 and 5,767,099, which are incorporated herein by reference.

Improved particles for the delivery of compounds is described in Ser.No. 09/680,690 and PCT/US00/27618, which are incorporated herein byreference. The subject matter described therein includes the use ofproviding particles that comprise co-stimulatory molecule ligands inorder to target cells that express the co-stimulatory molecules. Theparticles that comprise the co-stimulatory molecule ligands bind to andare taken up by cells that express the co-stimulatory molecules. Thus,compounds that are components of the particle are taken up by the cells.

The use of fusion proteins that comprise a portion of the HIV Vprprotein linked to biologically active non-HIV proteins is described inSer. No. 08/167,608 filed Dec. 15, 1993 and PCT/US94/02191 filed Jan.22, 1994, which are incorporated herein by reference. The subject matterdescribed therein sets forth the use of such fusion proteins to deliverbiologically active proteins using HIV particles, preferablynon-replicating HIV particles to deliver the fusion proteins. About 2400copies of the Vpr protein are packaged within the HIV particle. Byproviding fusion proteins with Vpr sequences that interact with the HIVparticle, 2400 copies of the fusion protein can be packaged within anHIV-derived particle. Packaging systems are described in each of thefollowing U.S. patents which are incorporated herein by reference: U.S.Pat. Nos. 5,932,467, 5,952,225, 5,932,467, 5,928,913, 5,919,676,5,912,338, 5,888,767, 5,872,005, 5,866,411, 5,843,723, 5,834,256,5,753,500, 5,739,018, 5,736,387, 5,723,287, 5,716,832, 5,710,037,5,693,531, 5,672,510, 5,665,577, 5,622,856, 5,587,308 and 5,585,254.

The delivery of heterologous gene sequences for expression includesthose delivered using particles as well as those which are free of suchparticles. For example, nucleic acid sequences may be included inviral-derived particles, liposomes or other complexes as well as in theform of free DNA delivered with or without co-agents. There are manywell known applications, such as vaccine and gene therapy, fordelivering nucleic acid molecules in expressible constructs to be takenup by cells and expressed. DNA vaccines are described in U.S. Pat. Nos.5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505,5,580,859, 5,703,055, 5,676,594, and the priority applications citedtherein, which are each incorporated herein by reference. In addition tothe delivery protocols described in those applications, alternativemethods of delivering DNA are described in U.S. Pat. Nos. 4,945,050 and5,036,006,which are both incorporated herein by reference. Examples ofattenuated live vaccines and those using recombinant vectors to deliverforeign antigens are described in U.S. Pat. Nos.: 4,722,848; 5,017,487;5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336;5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744;5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734; and5,482,713, which are each incorporated herein by reference.

There remains a need for improved particles for delivery of compounds tocells. There remains a need for improved expression systems for nucleicacid molecules delivered to cells.

SUMMARY OF THE INVENTION

The present invention provides three improvements which can be employedindependently or in combination in methods of delivering compounds to anindividual. One such improvement relates to methods of and compositionsfor particle based delivery of compounds. One such improvement relatesto methods of and compositions for particle based delivery of compoundsemploying viral particles, or particles derived therefrom. Oneimprovement can be applied to any method and composition for delivery ofDNA that is to be expressed in cells.

According to some aspects of the present invention, compositions andmethods are provided for the delivery of heterologous gene sequences forexpression in cells of an individual using gene constructs that includeexpression sequences from AAV, adenovirus or alpha viruses such as SFV.The expression sequences include the sequences responsible for bothintegration and expression and the constructs are free of the AAV,adenovirus or alpha viruses particles from which the expressionsequences are derived. In some embodiments, the constructs are includedin viral-derived particles, liposomes or other complexes as well as inthe form of free DNA delivered with or without co-agents. In someembodiments, the constructs include a packaging signal so that thenucleic acid molecule gets incorporated into a viral particle.

According to aspects of the invention, fusion proteins are providedwhich comprise HIV Vpr sequences and biologically active portionsselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins. The Vpr derived portion includes sequences which are requiredfor Vpr protein, and therefor the fusion protein, to be packaged withinan HIV derived particle. Optionally, the portions are linked by aprotease cleavage site.

Some aspects of the present invention arises from the discovery thatnon-cellular particle that comprises the compound and a FLT-3 ligand areparticularly useful to deliver a compound into a cell that expressesFLT-3 molecules. Accordingly, one aspect of the invention relates tomethods of introducing a compound into cells that expresses FLT-3molecules. The methods comprise contacting the cell with a non-cellularparticle that comprises the compound and a FLT-3 ligand. In someembodiments, the compound is a nucleic acid molecule or protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a construct used in the invention to produce a fusionprotein comprising the extracellular domain of LFT-3 ligand and thecytoplasmic domain of gp41.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Definitions

As used herein, the term “compound” is meant to refer to any moleculeincluding, but not limited to, a nucleic acid molecule such as DNA orRNA, or a proteinaceous molecule such as a peptide, polypeptide orprotein.

As used herein, the term “non-cellular particle” is meant to refer toany particulate structure except a cell.

As used herein, the phrase “cell that expresses costimulatory molecules”is meant to refer to any cell that express one or more costimulatorymolecules. Such cells are generally antigen presenting cells such asmacrophage, granulocyte, dendretic, monocyte, or B cells. Examples ofcostimulatory molecules are CD80, CD86, CD40, ICOSL, ICAM-1, 41BB,M-CSFR, FLT3, CCR-5, CCR-3, and CCR-2.

As used herein, the phrase “costimulatory ligand” is meant to refer to amolecule that specifically binds to a costimulatory molecule. Thecostimulatory ligand is a preferably protein, more preferably ananti-costimulatory molecule antibody, a natural ligand that is specificfor the costimulatory molecule, fragments thereof or a fusion proteinwhich includes a portion which specifically binds to a costimulatorymolecule. In some embodiments, the portion of a fusion protein whichspecifically binds to a costimulatory molecule is an anti-costimulatorymolecule antibody, a natural ligand that is specific for thecostimulatory molecule, or fragments thereof. The fusion protein mayfurther comprise portions which perform other functions.

As used herein, the term “natural ligand that is specific for thecostimulatory molecule” is meant to refer to the cellular proteinpresent on cells which binds to the costimulatory molecule present onanother cell. For example, CD28 and CTLA-4 are both natural ligands forCD80, CD28 is also a natural ligand for CD86, the natural ligand forCD40 is CD40L, the natural ligand for ICOSL is ICOS, the natural ligandfor ICAM-1 is LFA-3, the natural ligand for 41BB is 41BBL, the naturalligand for MCSFR is MCSF, the natural ligand for FT3 is FL3L, thenatural ligand for CCR2, CCR3 and CCR5 are MCP-3, and RANTES.

As used herein, the phrase “Flt-3 ligand” is meant to refer to amolecule that specifically binds to a Flt-3 molecule. The Flt-3 ligandis a preferably protein, more preferably an anti-Flt-3 moleculeantibody, the natural ligand that is specific for the Flt-3 molecule,fragments thereof or a fusion protein which includes a portion of thenatural ligand that is specific for the Flt-3 molecule whichspecifically binds to Flt-3. In some embodiments, the portion of afusion protein which specifically binds to Flt-3 is an anti-Flt-3antibody, the natural ligand that is specific for Flt-3, or fragmentsthereof. The fusion protein may further comprise portions which performother functions.

As used herein, the term “antibody” is meant to refer to antibodies, aswell as antibody fragments such as FAb and F(Ab)₂ fragments. Antibodiesmay, in some preferred embodiments, be monoclonal antibodies, primatizedantibodies or humanized antibodies. Antibodies may, in some preferredembodiments, be murine or human antibodies.

As used herein, the term “cationic amphiphile/DNA complex” is meant torefer to a complex arising from the mixture of DNA and one or morecationic amphiphiles.

As used herein the term “desired protein” is meant to refer to peptidesand protein encoded by gene constructs of the present invention whicheither act as target proteins for an immune response or as a therapeuticor compensating protein in gene therapy regimens.

As used herein, the term “genetic therapeutic” refers to apharmaceutical preparation that comprises a genetic construct thatcomprises a nucleotide sequence that encodes a therapeutic orcompensating protein.

As used herein, the term “therapeutic protein” is meant to refer toproteins whose presence confers a therapeutic benefit to the individual.

As used herein, the term “compensating protein” is meant to refer toproteins whose presence compensates for the absence of a fullyfunctioning endogenously produced protein due to an absent, defective,non-functioning or partially functioning endogenous gene.

As used herein, the term “biologically active portion” refers to theportion of a fusion protein derived from a biologically active proteinwhich retains and effects its biologically activity when taken up by acell.

FLT-3 Ligand

It has been discovered that particles that comprise FLT-3 ligand areparticularly useful to target cells and deliver compounds to the cellsusing such particles. It has been discovered that such particles areuseful in the absence of a fusion domain. According to a preferredembodiments, a fusion protein is provided that comprises a FLT-3 ligandportion linked to the transmembrane and cytoplasmic portions of HIVgp41. A construct for preparing a fusion protein according to thisaspect of the invention is set forth in FIG. 1. HIV env, derived fromHIV CCR5 has been found to be particularly useful. Particles assembledby, for example, packaging cell lines that express the fusion proteincomprise the FLT-3 ligand portion successfully bind to and are taken upby cells. In some preferred embodiments that include the fusion proteinthat comprises a FLT-3 ligand portion linked to the transmembrane andcytoplasmic portions of HIV gp41, the particles assembled by a packagingcell line that express the fusion protein and HIV env, preferably thatwhich is derived from HIV CCR5. The use of fusion proteins that comprisea FLT-3 ligand portion can be included in the particles for the deliveryof compounds described in PCT/US00/27618, particularly viral particlethat contain protein based stalks such as retroviruses, such aslentiviruses, particularly HIV-1 or HIV-2, and herpesviruses,particularly HSV-1 or HSV-2. In some preferred embodiments that includeherpes virus-derived particles, the fusion proteins may comprise theFLT-3 ligand portion linked to herpes virus viral protein gD, gB, gH orgL. Embodiments according to this aspect of the invention includeparticles in which the co-stimulatory ligand as described in Example 2is FLT-3 ligand.

Vpr Fusion Proteins

According to some aspects of the invention, fusion proteins are providedwhich in addition to Vpr sequences comprise biologically active portionsselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins. In some embodiments, the transcription factor is Tbet. Whenparticles that contain such fusion proteins infect cells, the action ofthe Tbet results in a shifting of the immune response to a Th1 response.Such particles are useful to improve immune responses induced byvaccines. In some embodiments, the transcription factor is Tgata. Whenparticles that contain such fusion proteins infect cells, the action ofthe Tgata results in a shifting of the immune response to a Th2response. Such particles are useful to treat patients with autoimmunediseases. Cytokines and chemokines can drive/modulate immune responses.Human proinflammatory cytokines include IL-1α, TNF-α and TNF-β, Th1cytokines include IL-2, IL-15, and IL-18, and Th2 cytokines includeIL-4, IL-5 and IL-10. GM-CSF is another factor which may be deliveredaccording to the invention. In some embodiments, the cytokine is IL-15.In some embodiments, the chemokine is RANTES. Fusion proteins cantransport proteins such as p70 or processing proteins such as Tap.

Fusion proteins preferably do not contain Vpr sequences not related topackaging. In particular, fusion proteins preferably do not contain Vprsequences that are involved in Vpr's cell cycle arrest/apoptosisinducing activity. Vpr sequences with their respective activities aredescribed in published PCT application PCT/US98/16890 and published PCTapplication PCT/US98/21432, both of which are incorporated herein byreference. The fusion protein may include a protease cleavage sitebetween the Vpr portion and the biologically active portion. An exampleof such a cleavage site is the cleavage site recognized by the HIVprotease. In addition to HIV-derived particles, the invention isenvisioned to include particles and fusion proteins based upon otherlentiviruses and lentivirus proteins. The production and use ofparticles according to this aspect of the invention is described inExamples 2 and 3 below. The invention also pertains to the use of freeVpr fusions to be delivered as drugs as well.

DNA Constructs

According to some aspects of the present invention, compositions andmethods are provided for the delivery of heterologous gene sequences forexpression in cells of an individual using gene constructs that includeexpression sequences from AAV, adenovirus or alpha viruses such as SFV.The expression sequences include the sequences responsible for bothintegration and expression and the constructs are free of the AAV,adenovirus or alpha viruses particles from which the expressionsequences are derived. The constructs may be included in viral-derivedparticles, liposomes or other complexes as well as in the form of freeDNA delivered with or without co-agents. The constructs are preferablyincluded in particles that will not cross react to the immune responsegenerated against a potent expression viral vectors. This will allowboosts while taking advantage of the high expression characteristics ofthe potent expression viral vectors. In preferred embodiments, theconstructs are part of retrovirus derived viral particles. The potentexpression sequences contain the regulatory sequences, such as the ITRsequences in the case of AAV, a gene of interest and a packaging signalso that the nucleic acid molecule gets incorporated into the viralparticle.

Combinations

According to the present invention, compositions include one, two or allthree of the above-described improvements. That is, in some embodiments,particles are provided with fusion proteins that include a FLT-3 ligandportion. In some embodiments, particles are provided with fusionproteins that include a FLT-3 ligand portion and a fusion protein thatincludes a Vpr portion and a biologically active portion selected fromthe group consisting of transcription factors, growth factors,cytokines, chemokines, transport proteins and processing proteins. Insome embodiments, particles are provided with fusion proteins thatinclude a FLT-3 ligand portion, a fusion protein that includes a Vprportion and a biologically active portion selected from the groupconsisting of transcription factors, growth factors, cytokines,chemokines, transport proteins and processing proteins, and a nucleicacid molecule that comprises expression sequences from AAV, adenovirusor alpha viruses. In some embodiments, particles are provided fusionproteins that include a Vpr portion and a biologically active portionselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins. In some embodiments, particles are provided with fusionproteins that include a Vpr portion and a biologically active portionselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins, and a nucleic acid molecule that comprises expressionsequences from AAV, adenovirus or alpha viruses. In some embodiments, anucleic acid molecules are provided that comprises expression sequencesfrom AAV, adenovirus or alpha viruses.

The methods of the present invention are useful in the fields of bothhuman and veterinary medicine. Accordingly, the present inventionrelates to genetic immunization of mammals, birds and fish. The methodsof the present invention can be particularly useful for mammalianspecies including human, bovine, ovine, porcine, equine, canine andfeline species.

The Examples set out below include representative examples of aspects ofthe present invention. The Examples are not meant to limit the scope ofthe invention but rather serve exemplary purposes. In addition, variousaspects of the invention can be summarized by the following description.However, this description is not meant to limit the scope of theinvention but rather to highlight various aspects of the invention. Onehaving ordinary skill in the art can readily appreciate additionalaspects and embodiments of the invention.

EXAMPLES Example 1

The following sequences identified by accession number and referencesare incorporated herein by reference.

-   VPR AJ404325 vpr, gag, pol, vif, vpu, env, and nef-   VPR AF316862 vif, vpr (Cameroon isolate)-   VPR AF325763 vif, vpr (South African isolate)-   macrophage colony-stimulating factor-   Accession No. AAA59572-   Cerretti, D. P. et al., Mol. Immunol. 25 (8), 761-770 (1988)-   Accession No. AAB51235-   Visvader, J. and Verma, I. M., Mol. Cell. Biol. 9 (3) 1336-1341    (1989)-   Accession No. P09603-   Wong et al., Science 235 (4795) 1504-1508 (1987)-   Cerretti et al., Mol. Immunol. 25 (8) 761-770 (1988)-   Kawasaki et al, Science 230 (4723) 291-296 (1985)-   chemokine (C-C motif) receptor 5-   Accession No. 4502639-   Raport, C. J. et al., J. Biol. Chem. 271 (29), 17161-17166 (1996)-   monocyte chemoattractant protein (MCP-3)-   Accession No. CAA50407-   Minty, A. et al., Eur. Cytokine Netw. 4 (2), 99-110 (1993)-   Accession No. AAC03538    pFLT3-   fms-related tyrosine kinase 3-   Accession No. 4758396-   Small, D. et al., Proc. Natl. Acad. Sci. U.S.A. 91, 459-463 (1994)-   Accession No. P36888-   Small et al., Proc. Natl. Acad. Sci. U.S.A. 91, 459-463 (1994)    pFLT3LG-   fms-related tyrosine kinase 3 ligand-   Accession No. 4503751    4-1BB-   Accession No. AAA53133-   Alderson, M. R. et al., Eur. J. Immunol. 24 (9), 2219-2227 (1994)    4-1BBL-   Accession No. P41273-   Alderson, M. R. et al., Eur. J. Immunol. 24 (9) 2219-2227 (1994)    RANTES-   Accession No. BAA76939-   Liu, H. et al., PNAS U.S.A. 96 (8), 4581-4585 (1999)-   Accession No. 1065018    CCR1/MIP1R-   Accession No. P32246-   Neote, K. et al., Cell 72 (3) 415-425 (1993)-   Gao, J. L. et al., J. Exp. Med. 177 (5) 1421-1427 (1993)-   Nomura, H. et al., Int. Immunol. 5 (10) 1239-1249 (1993)    CCR5-   Accession No. P56493-   Kuhmann, S. E. et al., J. Virol. 71 (11) 8642-8656 (1997)-   Murayama, Y. et al.    CCR2-   Accession No. P41597-   Charo, I. F. et al., PNAS, U.S.A. 91 (7) 2752-2756 (1994)-   Yamagami, S. et al., Biochem. Biophys. Res. Commun. 202 (2)    1156-1162 (1994)-   Wong, L. M. et al., J. Biol. Chem. 272 (2) 1038-1045 (1997)    CCR3-   Accession No. P51677-   Combadiere, C. et al., J. Biol. Chem. 270 (28) 16491-16494 (1995)-   Combadiere, C. et al., J. Biol. Chem. 270 30235 (1995)-   Dougherty, B. L. et al., J. Exp. Med. 183 (5) 2349-2354 (1996)    CD40 ligand-   Accession No. P29965-   Graf, D. et al., Eur. J. Immunol. 22 (12) 3191-3194 (1992)-   Hollenbaugh, D. et al., Embo. J. 11 (12) 4313-4321 (1992)-   Spriggs, M. K. et al., Cell 72 291-300 (1993)-   Spriggs, M. K. et al., J. Exp. Med. 176 (6) 1543-1550 (1992)-   Gauchat et al., Febs. Lett. 315 (3) 259-266 (1993)    CD86-   Accession No. 5901920-   Azuma et al., Nature 366 (6450) 76-79 (1993)-   Reeves et al., Mamm. Genome 8 (8) 581-582 (1997)    CD80-   Accession No. 4885123-   Selvakumar et al., Immunogenetic 36 (3) 175-181 (1992)-   Freeman et al., Blood 79 (2) 489-494 (1992)    CD40-   Accession No. 4507581-   Stamenkovic et al., Embo. J. 8 (5) 1403-1410 (1989)    LFA-3-   Accession No. BAA05922    ICAM1-   Accession No. AAB51145    CD28-   Accession No. 5453611-   Lee et al., J. Immunol. 145 (1) 344-352 (1990)

The nucleotide and amino acid sequences of human IL-1α are well knownand set forth in Telford, et al. (1986) Nucl. Acids Res. 14:9955-9963,Furutani, et al. (1985) Nucl. Acids Res. 14:3167-3179, March, et al.(1985) Nature 315:641-647, and accession code Swissprot PO1583, whichare each incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-2 are well known andset forth in Holbrook, et al. (1984) Proc. Natl. Acad. Sci. USA81:1634-1638, Fujita, et al. (1983) Proc. Natl. Acad. Sci. USA80:7437-7441, Fuse, et al. (1984) Nucl. Acids Res. 12:9323-9331,Taniguchi, et al. (1983) Nature 302:305-310, Maeda, et al. (1983)Biochem. Biophys. Res. Comm. 115:1040-1047, Devos, et al. (1983) Nucl.Acids Res. 11:4307-4323, and accession code Swissprot PO1585, which areeach incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-4 are well known andset forth in Arai, et al. (1989) J. Immunol. 142:274-282, Otsuka, et al.(1987) Nucl. Acids Res. 15:333-344, Yokota, et al. (1986) Proc. Natl.Acad. Sci. USA 83:5894-5898, Noma, et al. (1984) Nature 319:640-646,Lee, et al. (1986) Proc. Natl. Acad. Sci. USA 83:2061-2063, andaccession code Swissprot 05112 (the accession code for murine IL-4 isSwissprot 07750), which are each incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-5 are well known andset forth in Campbell, et al. (1987) Proc. Natl. Acad. Sci. USA84:6629-6633, Tanabe, et al. (1987) J. Biol. Chem. 262:16580-16584,Campbell, et al. (1988) Eur. J. Biochem. 174:345-352, Azuma, et al.(1986) Nucl. Acids Res. 14:9149-9158, Yokota, et al. (1986) Proc. Natl.Acad. Sci. USA 84:7388-7392, and accession code Swissprot PO5113, whichare each incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-10 are well knownand set forth in Viera, et al. (1991) Proc. Natl. Acad. Sci. USA88:1172-1176, and accession code Swissprot P22301.

The nucleotide and amino acid sequences of human IL-15 are well knownand set forth in Grabstein, et al. (1994) Science 264:965-968, andaccession code Swissprot U03099, which are each incorporated herein byreference.

The nucleotide and amino acid sequences of human IL-18 are well knownand set forth in Ushio, et al. (1996) J. Immunol. 156:4274-4279, andaccession code D49950, which are each incorporated herein by reference.

The nucleotide and amino acid sequences of human TNF-α are well knownand set forth in Pennica, (1984) Nature 312:724-729, and accession codeSwissprot PO1375, which are each incorporated herein by reference.

The nucleotide and amino acid sequences of human TNF-β are well knownand set forth in Gray, (1984) Nature 312:721-724, and accession codeSwissprot PO1374, which are each incorporated herein by reference.

T-bet.:

Susanne J. Szabo, et al., A Novel Transcription Factor, T-bet, DirectsTh1 Lineage Commitment, Cell 2000 100:655.

Shimon Sakaguchi, Regulatory T Cells: Key Controllers of ImmunologicSelf-Tolerance, Cell 2000 101:455.

The following provides the information from the Database, which providesinformation for both protein and gene sequences

-   1: AF241243 PubMed, Protein, Related Sequences, Taxonomy, OMIM Homo    sapiens T-cell-specific T-box transcription factor T-bet mRNA,    complete cds-   2: AF241242 PubMed, Protein, Related Sequences, Taxonomy, OMIM Mus    musculus T-cell-specific T-box transcription factor T-bet mRNA,    complete cds-   3: NM_(—)013351 PubMed, Protein, Related Sequences, Taxonomy, OMIM    Homo sapiens T-box 21 (TBX21), mRNA-   Tbet AF241243 human—also called T-cell specific T-box transcription    factor T-Gata.

Staal F J, et al, Transcriptional control of t lymphocytedifferentiation., Stem Cells. 2001;19(3):165-79.

Lee G R, et al., Regulation of IL-4 gene expression by distal regulatoryelements and GATA-3 at the chromatin level., Immunity. April2001;14(4):447-59.

Macaubas C, Holt P G. Regulation of cytokine production in T-cellresponses to inhalant allergen:GATA-3 expression distinguishes betweenTh1- and Th2-polarized immunity., Int Arch Allergy Immunol.January-March 2001;124(1-3):176-9.

Kitajima K, et al., A role of jumonji gene in proliferation but notdifferentiation of megakaryocyte lineage cells. Exp Hematol. April2001;29(4):507-14.

Toor A A, et al., T-cell factor-1 expression during human natural killercell development and in circulating CD56(+) bright natural killercells., Exp Hematol. April 2001;29(4):499-506.

Smits H H, et al., IL-12-induced reversal of human Th2 cells isaccompanied by full restoration of IL-12 responsiveness and loss ofGATA-3 expression., Eur J Immunol. April 2001;31(4):1055-65.

Crispino J D, et al., Proper coronary vascular development and heartmorphogenesis depend on interaction of GATA-4 with FOG cofactors., GenesDev. Apr. 1, 2001;15(7):839-44.

Christodoulopoulos P, et al., TH2 cytokine-associated transcriptionfactors in atopic and nonatopic asthma: evidence for differential signaltransducer and activator of transcription 6 expression. J Allergy ClinImmunol. April 2001;107(4):586-91.

Costa R H, et al., Transcription factors in mouse lung development andfunction. Am J Physiol Lung Cell Mol Physiol. May 2001;280(5):L823-38.

Grogan J L, et al., Early transcription and silencing of cytokine genesunderlie polarization of T helper cell subsets. Immunity. March2001;14(3):205-15.

Tilbrook P A, et al.,Maturation of erythroid cells and erythroleukemiadevelopment are affected by the kinase activity of Lyn. Cancer Res. Mar.15, 2001;61(6):2453-8.

Duan Z, et al., Role of NF-Y in in vivo regulation of the gamma-globingene. Mol Cell Biol. May 2001;21(9):3083-95.

Wright C E, et al., In vivo regulation of the beta-myosin heavy chaingene in hypertensive rodent heart. Am J Physiol Cell Physiol. May2001;280(5):C1262-76.

Ranganath S, Murphy K M. Structure and specificity of GATA proteins inTh2 development. Mol Cell Biol. April 2001;21(8):2716-25.

Tingvall T O, et al., The GATA factor Serpent is required for the onsetof the humoral immune response in Drosophila embryos. Proc Natl Acad SciU S A. Mar. 27, 2001;98(7):3884-8.

Das J, et al., A critical role for NF-kappa B in GATA3 expression andTH2 differentiation in allergic airway inflammation. Nat Immunol.January 2001;2(1):45-50.

Lantelme E, Kinetics of GATA-3 gene expression in early polarizing andcommitted human T cells. Immunology. February 2001;102(2):123-30.

Wong W K, et al., Regulation of human monoamine oxidase B gene by Sp1and Sp3. Mol Pharmacol. April 2001;59(4):852-9.

Gilleard J S, McGhee J D. Activation of hypodermal differentiation inthe Caenorhabditis elegans embryo by GATA transcription factors ELT-1and ELT-3. Mol Cell Biol. April 2001;21(7):2533-44.

Tremblay J J, Viger R S. Nuclear receptor Dax-1 represses thetranscriptional cooperation between GATA-4 and SF-1 in Sertoli cells.,Biol Reprod. April 2001;64(4):1191-9.

-   1: AE006819 Protein, Related Sequences, Genome, Taxonomy Sulfolobus    solfataricus section 178 of 272 of the complete genome-   2: AL590734 PubMed, Protein, Taxonomy Leishmania major chromosome 13    clone PAC P883 strain Friedlin-   3: AR106378 Sequence 10 from patent U.S. Pat. No. 6,107,034-   4: AR106377 Sequence 9 from patent U.S. Pat. No. 6,107,034-   5: AR106376 Sequence 8 from patent U.S. Pat. No. 6,107,034-   6: AR106375 Sequence 7 from patent U.S. Pat. No. 6,107,034-   7: AR106374 Sequence 6 from patent U.S. Pat. No. 6,107,034-   8: AR106373 Sequence 5 from patent U.S. Pat. No. 6,107,034-   9: AR106372 Sequence 4 from patent U.S. Pat. No. 6,107,034-   10: AR106371 Sequence 3 from patent U.S. Pat. No. 6,107,034-   11: AR106370 Sequence 2 from patent U.S. Pat. No. 6,107,034-   12: AR106369 Sequence 1 from patent U.S. Pat. No. 6,107,034-   13: AY024364Protein, Taxonomy Rattus norvegicus GATA-3 mRNA,    complete cds-   14: NM_(—)008091 PubMed, Protein, Related Sequences, Taxonomy Mus    musculus GATA-binding protein 3 (Gata3), mRNA-   15: NM_(—)002051 PubMed, Protein, Related Sequences, Taxonomy, OMIM    Homo sapiens GATA-binding protein 3 (GATA3), mRNA-   16: BB509565 RIKEN full-length enriched, 10 days lactation, adult    female mammary gland Mus musculus cDNA clone D730025A17 similar to    X55123 Mouse mRNA for GATA-3 transcription factor, mRNA sequence-   17: BB508018RIKEN full-length enriched, 10 days lactation, adult    female mammary gland Mus musculus cDNA clone D730015M23 similar to    X55123 Mouse mRNA for GATA-3 transcription factor, mRNA sequence-   18: BB501281 RIKEN full-length enriched, 0 day neonate kidney Mus    musculus cDNA clone D630034A10 3′ similar to X55123 Mouse mRNA for    GATA-3 transcription factor, mRNA sequence-   19: BB497281 RIKEN full-length enriched, 0 day neonate kidney Mus    musculus cDNA clone D630008B05 3′ similar to X55123 Mouse mRNA for    GATA-3 transcription factor, mRNA sequence

20: BB121131 RIKEN full-length enriched, adult male urinary bladder Musmusculus cDNA clone 9530080K05 3′ similar to X55123 Mouse mRNA forGATA-3 transcription factor, mRNA sequence Tgata XM 010214 human--GATA1Tgata NM002049 human--GATA1 Tgata NM005257 human--GATA6

Example 2

The present invention relates to methods of introducing compounds intocells that express costimulatory molecules, and to non-cellularparticles useful in such methods. According to the methods of thepresent invention, cells that express costimulatory molecules arecontacted with non-cellular particles that comprise a compound incombination with a costimulatory ligand. The costimulatory ligandcomponent of the particle specifically target the cells that expresscostimulatory molecules. The particles bind to the cells and are takenup by them, thus delivering the compound into the cell.

According to some aspects of the present invention, methods ofimmunizing individuals are provided. Such methods comprise the step ofadministering to tissue of the individual at a site on the individual'sbody, a non-cellular particle that comprises an immunogenic protein or anucleic acid molecule that encodes an immunogenic protein. The particleadditionally comprises costimulatory ligand. The particles bind to thecells and are taken them, thus delivering the immunogenic protein or anucleic acid molecule that encodes an immunogenic protein into the cell.An immune response is generated against the immunogenic proteindelivered to the cell or against the expression product of a nucleicacid molecule which encodes an immunogenic protein and which is taken upby and expressed in the cell.

The present invention maybe used to immunize an individual against allpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites.

Another aspect of the present invention provides a method of conferringa broad based protective immune response against hyperproliferatingcells that are characteristic in hyperproliferative diseases and to amethod of treating individuals suffering from hyperproliferativediseases. As used herein, the term “hyperproliferative diseases” ismeant to refer to those diseases and disorders characterized byhyperproliferation of cells. Examples of hyperproliferative diseasesinclude all forms of cancer and psoriasis. The present inventionprovides a method of treating individuals suffering fromhyperproliferative diseases. In such methods, the compound provides atarget protein against which an immune response that will be specificfor proteins expressed by hyperproliferating cells. While the presentinvention may be used to immunize an individual against one or more ofseveral forms of cancer, the present invention is particularly useful toprophylactically immunize an individual who is predisposed to develop aparticular cancer or who has had cancer and is therefore susceptible toa relapse. Developments in genetics and technology as well asepidemiology allow for the determination of probability and riskassessment for the development of cancer in individual. Using geneticscreening and/or family health histories, it is possible to predict theprobability a particular individual has for developing any one ofseveral types of cancer. Similarly, those individuals who have alreadydeveloped cancer and who have been treated to remove the cancer or areotherwise in remission are particularly susceptible to relapse andreoccurrence. As part of a treatment regimen, such individuals can beimmunized against the cancer that they have been diagnosed as having hadin order to combat a recurrence. Thus, once it is known that anindividual has had a type of cancer and is at risk of a relapse, theycan be immunized in order to prepare their immune system to combat anyfuture appearance of the cancer.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies.

According to some aspects of the present invention, methods ofdelivering therapeutic compounds to individuals are provided. Accordingto such methods, the compound is a therapeutic compound. In someembodiments, the compound is therapeutic protein or a nucleic acidmolecule that encodes a therapeutic protein. The methods comprise thestep of administering to tissue of the individual at a site on theindividual's body, a non-cellular particle that comprises an therapeuticprotein or a nucleic acid molecule that encodes an therapeutic protein.The particle additionally comprises costimulatory ligand. The particlesbind to the cells and are taken them, thus delivering the therapeuticprotein or a nucleic acid molecule that encodes an therapeutic proteininto the cell. The therapeutic protein is thus delivered directly to thecell or is produced in the cell by the of the nucleic acid moleculewhich encodes it and is taken up in the cell.

Some aspects of the present invention relate to gene therapy; that is,to compositions for and methods of introducing nucleic acid moleculesinto the cells of an individual exogenous copies of genes which eithercorrespond to defective, missing, non-functioning or partiallyfunctioning genes in the individual or which encode therapeuticproteins, i.e. proteins whose presence in the individual will eliminatea deficiency in the individual and/or whose presence will provide atherapeutic effect on the individual thereby providing a means ofdelivering the protein by an alternative means from proteinadministration.

Compounds

Compounds which can be delivered to cells by the methods of theinvention may be any molecule. In some embodiments, the compound is anucleic acid molecule such as DNA or RNA. In some embodiments, thecompound is a proteinaceous molecule such as a peptide, polypeptide orprotein.

In some embodiments, the compound is a protein molecule. In someembodiments, the compound is an immunogenic protein. In someembodiments, the compound is a non-immunogenic protein molecule.

Examples of immunogenic proteins includes pathogen antigens,proteinaceous allergans, immunogenic proteins associated with cancercells, and immunogenic proteins associated with cells involved inautoimmune diseases.

Pathogen antigens may be derived from all pathogens such as viruses,prokaryote and pathogenic eukaryotic organisms such as unicellularpathogenic organisms and multicellular parasites. The present inventionis particularly useful to immunize an individual against those pathogenswhich infect cells and which are not encapsulated such as viruses, andprokaryote such as gonorrhea, listeria and shigella. In addition, thepresent invention is also useful to immunize an individual againstprotozoan pathogens which include a stage in the life cycle where theyare intracellular pathogens. As used herein, the term “intracellularpathogen” is meant to refer to a virus or pathogenic organism that, atleast part of its reproductive or life cycle, exists within a host celland therein produces or causes to be produced, pathogen proteins. Table1 provides a listing of some of the viral families and genera for whichvaccines according to the present invention can be made. DNA constructsthat comprise DNA sequences which encode the peptides that comprise atleast an epitope identical or substantially similar to an epitopedisplayed on a pathogen antigen such as those antigens listed on thetables are useful in vaccines. Moreover, the present invention is alsouseful to immunize an individual against other pathogens includingprokaryotic and eukaryotic protozoan pathogens as well as multicellularparasites such as those listed on Table 2. Tables 1 and 2 include listsof some of the pathogenic agents and organisms for which geneticvaccines can be prepared to protect an individual from infection bythem. In some preferred embodiments, the methods of immunizing anindividual against a pathogen are directed against HIV, HTLV or HBV.

As used herein, the term “hyperproliferative-associated protein” ismeant to refer to proteins that are associated with a hyperproliferativedisease. To immunize against hyperproliferative diseases, a“hyperproliferative-associated protein” or a genetic construct thatincludes a nucleotide sequence which encodes a protein that isassociated with a hyperproliferative disease is included as the compoundin the particle administered to an individual. In order for thehyperproliferative-associated protein to be an effective immunogenictarget, it must be a protein that is produced exclusively or at higherlevels in hyperproliferative cells as compared to normal cells. Targetantigens include such proteins, fragments thereof and peptides whichcomprise at least an epitope found on such proteins. In some cases, ahyperproliferative-associated protein is the product of a mutation of agene that encodes a protein. The mutated gene encodes a protein which isnearly identical to the normal protein except it has a slightlydifferent amino acid sequence which results in a different epitope notfound on the normal protein. Such target proteins include those whichare proteins encoded by oncogenes such as myb, myc, fyn, and thetranslocation gene bcr/abl, ras, src, P53, neu, trk and EGRF. Inaddition to oncogene products as target antigens, target proteins foranti-cancer treatments and protective regimens include variable regionsof antibodies made by B cell lymphomas and variable regions of T cellreceptors of T cell lymphomas which, in some embodiments, are also usedtarget antigens for autoimmune disease. Other tumor-associated proteinscan be used as target proteins such as proteins which are found athigher levels in tumor cells including the protein recognized bymonoclonal antibody 17-1A and folate binding proteins.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vβ-17 and Vα-17. Thus, vaccination with a particlethat contains as the compound one of these proteins or a DNA constructthat encodes at least one of these proteins will result in thegeneration of an immune response that will target T cells involved inRA. See: Howell, M. D., et al., 1991 Proc. Natl. Acad. Sci. USA88:10921-10925; Paliard, X., et al., 1991 Science 253:325-329; Williams,W. V., et al., 1992 J. Clin. Invest. 90:326-333; each of which isincorporated herein by reference.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vα-10.Thus, vaccination with a particle that contains as the compound one ofthese proteins or a DNA construct that encodes at least one of theseproteins will result in the generation of an immune response that willtarget T cells involved in MS. See: Wucherpfennig, K. W., et al., 1990Science 248:1016-1019; Oksenberg, J. R., et al., 1990 Nature345:344-346; each of which is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a particle that contains as the compoundone of these proteins or a DNA construct that encodes at least one ofthese proteins will result in the generation of an immune response thatwill target T cells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Particles useful to immunizeagainst the disease can be prepared using this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Particles useful to immunize against such diseasescan be prepared using this information.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes the variableregion of those antibodies or DNA constructs that encode the variableregion of such anti-DNA antibodies found in the sera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.USA 87:1066, which is incorporated herein by reference.

In some embodiments the compound in the particle is a non-immunogenicprotein which may serve as replacement protein in individuals sufferingfrom diseases associated with defective, missing or non-functioninggenes. The non-immunogenic proteins may alternatively be therapeuticproteins. In some embodiments the compound in the particle is a nucleicacid molecule which serves as: 1) replacement copies of defective,missing or non-functioning genes; 2) genetic templates for therapeuticproteins; 3) genetic templates for antisense molecules; or 4) genetictemplates for ribozymes. In the case of nucleic acid molecules whichencode proteins, the nucleic acid molecules preferably comprise thenecessary regulatory sequences for transcription and translation in thecells of the animal. In the case of nucleic acid molecules which serveas templates for antisense molecules and ribozymes, such nucleic acidmolecules are preferably linked to regulatory elements necessary forproduction of sufficient copies of the antisense and ribozyme moleculesencoded thereby respectively. The nucleic acid molecules are free fromretroviral particles and preferably provided as DNA in the form ofplasmids.

In some of the embodiments of the invention that relate to gene therapy,the gene constructs contain either compensating genes or genes thatencode therapeutic proteins. Examples of compensating genes include agene which encodes dystrophin or a functional fragment, a gene tocompensate for the defective gene in patients suffering from cysticfibrosis, an insulin, a gene to compensate for the defective gene inpatients suffering from ADA, and a gene encoding Factor VIII.Additionally, genetic constructs which encode antibodies, such as singlechain antibody components which specifically bind to toxic substances,can be administered. In some embodiments, antibodies expressed in suchcells can be secreted. In some preferred embodiments, the dystrophingene is provided as part of a mini-gene and used to treat individualssuffering from muscular dystrophy. In some preferred embodiments, amini-gene which contains coding sequence for a partial dystrophinprotein is provided. Dystrophin abnormalities are responsible for boththe milder Becker's Muscular Dystrophy (BMD) and the severe Duchenne'sMuscular Dystrophy (DMD). In BMD dystrophin is made, but it is abnormalin either size and/or amount. The patient is mild to moderately weak. InDMD no protein is made and the patient is chair-bound by age 13 andusually dies by age 20. In some patients, particularly those sufferingfrom BMD, partial dystrophin protein produced by expression of amini-gene delivered according to the present invention can provideimproved muscle function.

Examples of therapeutic proteins include the proteins themselves and thegenes which encodes active proteins such as cytokines, growth factors,chemokines as well as toxins. In some embodiments, the protein iserythropoietin, interferon, LDL receptor, GM-CSF, IL-2, IL-4 or TNF.Therapeutic proteins or nucleic acid molecules that encode therapeuticproteins may be included in particles as a compound to be delivered tocells. Therapeutic proteins that are toxins cor otherwise toxic orcytostatic to the cell are useful for example when delivered to antigenpresenting cells in patients with lymphoproliferative diseases. Inaddition to toxins, other anti-proliferative proteins are antibodies,HIV Vpr and TGFβ. Therapeutic proteins that expand APC numbers includegrowth factors such as EPO, CSF and GCSF. Proteins which modulate immuneresponses may be delivered to cells in this manner in order to modulateimmune responses in an individual.

Antisense molecules and ribozymes may also be delivered to the cells ofan individual by introducing genetic material which acts as a templatefor copies of such active agents. These agents inactivate or otherwiseinterfere with the expression of genes that encode proteins whosepresence is undesirable. Constructs which contain sequences that encodeantisense molecules can be used to inhibit or prevent production ofproteins within cells. Thus, production proteins such as oncogeneproducts can be eliminated or reduced. Similarly, ribozymes can disruptgene expression by selectively destroying messenger RNA before it istranslated into protein. in some embodiments, cells are treatedaccording tot he invention using constructs that encode antisense orribozymes as part of a therapeutic regimen which involves administrationof other therapeutics and procedures. Gene constructs encoding antisensemolecules and ribozymes use similar vectors as those which are used whenprotein production is desired except that the coding sequence does notcontain a start codon to initiate translation of RNA into protein.

Ribozymes are catalytic RNAs which are capable of self-cleavage orcleavage of another RNA molecule. Several different types of ribozymes,such as hammerhead, hairpin, Tetrahymena group I intron, ahead, andRNase P are known in the art. (S. Edgington, Biotechnology 1992 10,256-262.) Hammerhead ribozymes have a catalytic site which has beenmapped to a core of less than 40 nucleotides. Several ribozymes in plantviroids and satellite RNAs share a common secondary structure andcertain conserved nucleotides. Although these ribozymes naturally serveas their own substrate, the enzyme domain can be targeted to another RNAsubstrate through base-pairing with sequences flanking the conservedcleavage site. This ability to custom design ribozymes has allowed themto be used for sequence-specific RNA cleavage (G. Paolella et al., EMBO1992, 1913-1919.) It will therefore be within the scope of one skilledin the art to use different catalytic sequences from various types ofribozymes, such as the hammerhead catalytic sequence and design them inthe manner disclosed herein. Ribozymes can be designed against a varietyof targets including pathogen nucleotide sequences and oncogenicsequences. Certain preferred embodiments of the invention includesufficient complementarity to specifically target the abl-bcr fusiontranscript while maintaining efficiency of the cleavage reaction.

Peptides, polypeptides and protein may be isolated from natural sources,synthesized or produced by recombinant methodology.

Recombinant expression vectors that comprises a nucleotide sequence thatencodes proteins of the invention can be produced routinely. As usedherein, the term “recombinant expression vector” is meant to refer to aplasmid, phage, viral particle or other vector which, when introducedinto an appropriate host, contains the necessary genetic elements todirect expression of a coding sequence. One having ordinary skill in theart can isolate or synthesize a nucleic acid molecule that encodes aprotein of the invention and insert it into an expression vector usingstandard techniques and readily available starting materials. The codingsequence is operably linked to the necessary regulatory sequences.Expression vectors are well known and readily available. Examples ofexpression vectors include plasmids, phages, viral vectors and othernucleic acid molecules or nucleic acid molecule containing vehiclesuseful to transform host cells and facilitate expression of codingsequences. The recombinant expression vectors of the invention areuseful for transforming hosts.

Host cells that comprise the recombinant expression vector can be usedto produce the protein. Host cells for use in well known recombinantexpression systems for production of proteins are well known and readilyavailable. Examples of host cells include bacteria cells such as E.coli, yeast cells such as S. cerevisiae, insect cells such as S.frugiperda, non-human mammalian tissue culture cells chinese hamsterovary (CHO) cells and human tissue culture cells such as HeLa cells.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert DNA molecules into acommercially available expression vector for use in well knownexpression systems. For example, the commercially available plasmidpSE420 (Invitrogen, San Diego, Calif.) may be used for production of aCD80ΔC mutant protein in E. coli. The commercially available plasmidpYES2 (Invitrogen, San Diego, Calif.) may, for example, be used forproduction in S. cerevisiae strains of yeast. The commercially availableMAXBAC™ complete baculovirus expression system (Invitrogen, San Diego,Calif.) may, for example, be used for production in insect cells. Thecommercially available plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego,Calif.) may, for example, be used for production in mammalian cells suchas Chinese Hamster Ovary cells. One having ordinary skill in the art canuse these commercial expression vectors and systems or others to produceproteins of the invention using routine techniques and readily availablestarting materials. (See e.g., Sambrook et al., Molecular Cloning aLaboratory Manual, Second Ed. Cold Spring Harbor Press (1989) which isincorporated herein by reference.) Thus, the desired proteins can beprepared in both prokaryotic and eukaryotic systems, resulting in aspectrum of processed forms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989).

The expression vector including the DNA that encodes a protein is usedto transform the compatible host which is then cultured and maintainedunder conditions wherein expression of the foreign DNA takes place. Theprotein of the invention thus produced is recovered from the culture,either by lysing the cells or from the culture medium as appropriate andknown to those in the art. One having ordinary skill in the art can,using well known techniques, isolate the protein of the invention thatis produced using such expression systems. The methods of purifyingproteins of the invention from natural sources using antibodies whichspecifically bind to such protein are routine as is the methods ofgenerating such antibodies (See: Harlow, E. and Lane, E., Antibodies: ALaboratory Manual, 1988, Cold Spring Harbor Laboratory Press which isincorporated herein by reference.). Such antibodies may be used topurifying proteins produced by recombinant DNA methodology or naturalsources.

Examples of genetic constructs include coding sequences which encode aprotein of the invention and which are operably linked to a promoterthat is functional in the cell line into which the constructs aretransfected. Examples of constitutive promoters include promoters fromcytomegalovirus or SV40. Examples of inducible promoters include mousemammary leukemia virus or metallothionein promoters. Those havingordinary skill in the art can readily produce genetic constructs usefulfor transfecting with cells with DNA that encodes proteins of theinvention from readily available starting materials. Such geneconstructs are useful for the production of proteins of the invention.

In addition to producing proteins of the invention by recombinanttechniques, automated peptide synthesizers may also be employed toproduce proteins of the invention. Such techniques are well known tothose having ordinary skill in the art and are useful if derivativeswhich have substitutions not provided for in DNA-encoded proteinproduction.

The proteins of the invention may be prepared by any of the followingknown techniques. Conveniently, the proteins of the invention may beprepared using the solid-phase synthetic technique initially describedby Merrifield, in J. Am. Chem. Soc., 15:2149-2154 (1963) which isincorporated herein by reference. Other protein synthesis techniques maybe found, for example, in M. Bodanszky et al., (1976) Peptide Synthesis,John Wiley & Sons, 2d Ed. which is incorporated herein by reference;Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, p.295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985)which is incorporated herein by reference; as well as other referenceworks known to those skilled in the art. A summary of synthesistechniques may be found in J. Stuart and J. D. Young, Solid PhasePeptide Synthelia, Pierce Chemical Company, Rockford, Ill. (1984) whichis incorporated herein by reference. Synthesis by solution methods mayalso be used, as described in The Proteins, Vol. II, 3d Ed., p. 105-237,Neurath, H. et al., Eds., Academic Press, New York, N.Y. (1976) which isincorporated herein by reference. Appropriate protective groups for usein such syntheses will be found in the above texts, as well as in J. F.W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, NewYork, N.Y. (1973) which is incorporated herein by reference.

In general, these synthetic methods involve the sequential addition ofone or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively-removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptide of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

In some embodiments, proteins may be produced in transgenic animals.Transgenic non-human mammals useful to produce recombinant proteins arewell known as are the expression vectors necessary and the techniquesfor generating transgenic animals. Generally, the transgenic animalcomprises a recombinant expression vector in which the nucleotidesequence that encodes a protein. is operably linked to a mammary cellspecific promoter whereby the coding sequence is only expressed inmammary cells and the recombinant protein so expressed is recovered fromthe animal's milk. One having ordinary skill in the art using standardtechniques, such as those taught in U.S. Pat. No. 4,873,191 issued Oct.10, 1989 to Wagner and U.S. Pat. No.4,736,866 issued Apr. 12, 1988 toLeder, both of which are incorporated herein by reference, can producetransgenic animals which produce a desired protein. Preferred animalsare goats, and rodents, particularly rats and mice.

In some embodiments, the compound is a nucleic molecule, preferably aDNA molecule. In some embodiments, the nucleic acid molecule is anantisense molecule, which when taken up by the cell, prevents orotherwise inhibits expression of a gene in the cell. In someembodiments, the nucleic acid molecule is a gene construct whichcontains a coding sequence operably linked to regulatory elementsnecessary for gene expression of a nucleic acid molecule in the cell.

In addition to a coding sequence, the elements of a gene constructinclude a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers are often required forgene expression of the sequence that encodes the protein. It isnecessary that these elements be operable linked to the sequence thatencodes the desired proteins and that the regulatory elements areoperably in the individual to whom they are administered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to human and bovine growth hormonepolyadenylation signals, SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs of the invention can be provided with mammalianorigin of replication in order to maintain the constructextrachromosomally and produce multiple copies of the construct in thecell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.)contain the Epstein Barr virus origin of replication and nuclear antigenEBNA-1 coding region which produces high copy episomal replicationwithout integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered-which include nucleotidesequences that encode immunogenic proteins, and additionally, genes forproteins which further enhance the immune response against such targetproteins. Examples of such genes are those which encode cytokines andlymphokines such as α-interferon, gamma-interferon, platelet derivedgrowth factor (PDGF), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF),IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and B7.2.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells intowhich the construct is to be administered. Moreover, codons may beselected which are most efficiently transcribed in the cell. One havingordinary skill in the art can produce DNA constructs which arefunctional in the cells.

In some embodiments, the compound is a DNA molecule. In someembodiments, the compounds is a DNA molecule that is a plasmid. In someembodiments, the compound is a DNA molecule that comprises a nucleotidesequences that encodes a protein operably linked to regulatory elementsfunctional in the cell. In some embodiments, the compound is a DNAmolecule that comprises an immunogenic protein operably linked toregulatory elements functional in the cell. In some embodiments, thecompound is a DNA molecule that comprises an immunogenic pathogenprotein operably linked to regulatory elements functional in the cell.In some embodiments, the compound is a DNA molecule that comprises anon-immunogenic protein operably linked to regulatory elementsfunctional in the cell.

DNA vaccines are described in U.S. Pat. No. 5,593,972, U.S. Pat. No.5,589,466, PCT/US90/01515, PCT/US93/02338, PCT/US93/048131, andPCT/US94/00899, and the priority applications cited therein each of thepatents and published patent applications, which are each incorporatedherein by reference. In addition to the delivery protocols described inthose applications, alternative methods of delivering DNA are describedin U.S. Pat. Nos. 4,945,050 and 5,036,006, which are both incorporatedherein by reference.

According to some embodiments, the compounds is a protein which includesviral sequences which function to package the compound in the viralparticle. In some embodiments, the viral sequences are viral proteins.In some embodiments, the viral sequences are fragments of viral proteinswhich retain their ability to complex with other viral proteins in theassembly of viral particles. In some embodiments, the particle is an HIVparticle and the compound is a fusion protein which includes sequencesof the HIV Vpr protein. The fusion protein which includes sequences ofthe HIV Vpr protein are packaged in the HIV particle.

Non-Cellular Particles

The non-cellular particles according to these aspects of the inventioninclude, but are not limited to, viral particles, protein complexes,liposomes and cationic amphiphile/DNA complexes. According to theinvention, such non-cellular particles include a costimulatory moleculeligand or fusion protein which includes a costimulatory molecule ligandportion in order to target the particles to the cells which displaycostimulatory molecules which bind to the costimulatory molecule ligandor fusion protein displayed by the particle. It has been discovered thatin addition to delivering the particles to the cells for localization tocells that display the costimulatory molecule, the particles accordingto the present invention which are delivered to and localized to cellsthat display the costimulatory molecule are taken up by the cells.

According to some embodiments of the invention, the particles are viralparticles. In preferred embodiments, the particles are non-replicatingviral particles. U.S. Pat. No. 5,714,316, which is incorporated hereinby reference, describes the design and production of viral particleswhich display heterologous protein sequences on the viral particleenvelope. The present invention provides an improvement to thistechnology by providing as the heterologous protein, either acostimulatory molecule ligand or fusion protein which includes acostimulatory molecule ligand portion. In some embodiments, theparticles are HIV, HIV, HCV or Papillomavirus particles, preferablynon-replicating.

Examples of viral particles according to the invention includenon-replicating HIV particles, adenovirus particles, and adenovirus-likeparticles. Non-replicating viruses are produced using packaging celllines. Packaging systems are described in each of the following U.S.patents which are incorporated herein by reference: U.S. Pat. Nos.5,932,467, 5,952,225, 5,932,467, 5,928,913, 5,919,676, 5,912,338,5,888,767, 5,872,005, 5,866,411, 5,843,723, 5,834,256, 5,753,500,5,739,018, 5,736,387, 5,723,287, 5,716,832, 5,710,037, 5,693,531,5,672,510, 5,665,577, 5,622,856, 5,587,308 and 5,585,254.

According to some embodiments, the particles are attenuated vaccineswhich are improved by providing them with costimulatory ligands totarget cells that express costimulatory molecules. Any of thecommercially available attenuated vaccines including those currentlybeing investigated such as those undergoing preclinical or clinicalpremarket testing may be improved by the present invention.

According to some embodiments of the invention, the particles areliposome particles. U.S. Pat. Nos. 4,873,089, 5,227,470 and 5,258,499,which are incorporated herein by reference, describe methods ofpreparing liposomes that contain proteins displayed on their surfaces inorder to target the liposomes to a cell with a cellular protein on itssurface that specifically binds to the protein on the surface of theliposome. The present invention provides a specific application of thistechnology by providing as the receptor ligand, either a costimulatorymolecule ligand or fusion protein which includes a costimulatorymolecule ligand portion. Liposomes include positive charged, negativecharged and neutral liposomes.

According to some embodiments of the invention, the particles arecationic amphiphile/DNA complexes. U.S. Pat. Nos. 5,837,533, 5,459,127and Behr, J. P., et al. (1989) Proc. Natl. Acad. Sci. USA 86:6982-6986,which are each incorporated herein by reference, describe the design andproduction of receptor targeted cationic amphiphile/DNA complexes inwhich positively charged lipophilic compounds are provided with receptorligands. The cationic amphiphilic compounds contain receptor ligandmoieties which are displayed on the surface of complexes formed when thecationic amphiphile is mixed with DNA. Such teachings may also beapplied to cationic lipid/DNA complexes such as those described in U.S.Pat. Nos. 5,955,365, 5,948,767, 5,945,400, 5,939,401, 5,935,936,5,932,241, 5,925,628, 5,916,803, 5,910,488, 5,908,635, 5,891,468,5,885,613, 5,830,430, 5,827,703, 5,783,565 and 5,767,099, which areincorporated herein by reference. In some embodiments, receptor ligandmoieties are not linked to any molecule or are linked to neutral lipidswhich are mixed with the cationic amphiphile and DNA and incorporatedinto any complexes formed thereby. According to the present invention,cationic amphiphile/DNA are provided with receptor ligands that arecostimulatory molecule ligands. Such complexes are targeted to cellsthat display costimulatory molecules. The complexes localize to and aretaken up by the cells.

According to some embodiments of the invention, the particles areprotein complexes which comprise two or more protein molecules. Theprotein complexes comprise a compound to be delivered and acostimulatory ligand.

Cells

The present invention provides methods of delivering compounds to acells that expresses costimulatory molecules. Typically, cells thatexpress costimulatory molecules are antigen presenting cells. In someembodiments, the method is directed at delivering compounds to a cellthat expresses costimulatory molecules that is a dendretic cell. In someembodiments, the method is directed at delivering compounds to a cellthat expresses costimulatory molecules that is a macrophage cell.

By delivering immunogens to these cells, immune responses can begenerated. By delivering therapeutic proteins which modulate immuneresponses to these cells, immune responses can be modified. Bydelivering toxins to these cells, immune responses can be reduced. Bydelivering growth factors to these cells, immune responses can beenhanced.

Ligands

The costimulatory ligand is a molecule that specifically binds to acostimulatory molecule. In some embodiments, the costimulatory ligand isa protein, preferably an anti-costimulatory molecule antibody, a naturalligand that is specific for the costimulatory molecule or a fusionprotein which comprises either an anti-costimulatory molecule antibody,natural ligand or functional fragment thereof.

Anti-costimulatory molecule antibody can be prepared from readilyavailable starting materials using routine techniques. Antibodiesagainst CD80, CD86, CD40, ICOSL, ICAM-1, 41BB, MCSFR, FLT3, CCR-5, CCR-3and CCR-2 may be used in particles of the invention in order to targetthe particles to cells expressing CD80, CD86, CD40, ICOSL, ICAM-1, 41BB,MCSFR, FLT3, CCR-5, CCR-3 and CCR-2 respectively.

Alternatively, natural ligands of CD80, CD86, CD40, ICOSL, ICAM-1, 41BB,MCSFR, FT3, CCR-5, CCR-3 and CCR-2 may be provided as costimulatoryligands in order to target the particles to cells expressing CD80, CD86,CD40, ICOSL, ICAM-1, 41BB, MCSFR, FLT3, CCR-5, CCR-3 and CCR-2respectively. The natural ligands include: CD28 and CTLA-4 which areboth natural ligands for CD80; CD28, a natural ligand for CD86; CD40L,the natural ligand for CD40; ICOS, the natural ligand for ICOSL; LFA-3the natural ligand for ICAM-1; 41BBL, the natural ligand for 41BB; MCSF,the natural ligand for MCSFR; FL3L, the natural ligand for FLT3; MCP3and RANTES, the natural ligand for CCR-5, CCR-3 and CCR-2. The methodsfor preparing or otherwise obtaining these proteins are well known.

In some embodiments, the costimulatory ligand is a fusion protein whichincludes a costimulatory ligand portion. In some embodiments, thecostimulatory ligand is portion is an anti-costimulatory moleculeantibody. In some embodiments, the costimulatory ligand is portion is acomplete natural costimulatory ligand molecule. In some embodiments, thecostimulatory ligand portion is a fragment of a natural costimulatoryligand molecule which retains its ability to bind to a costimulatorymolecule.

In some embodiments the costimulatory ligand is a fusion protein whichcomprises amino acid sequences which function in particle assembly orare involved in localizing the fusion protein on the particle. Forexample, in some embodiments the fusion protein further comprises viralprotein sequences which function in particle assembly such that thefusion protein becomes part of a viral particle. In some embodiments,the costimulatory ligand is a fusion protein that includes acostimulatory ligand portion and a viral protein portion. In someembodiments, the viral protein portion is a complete viral proteinmolecule. In some embodiments, the viral protein portion is a fragmentof a viral protein. In some embodiments, the viral protein portion is afragment of a viral protein that comprise the internal domain andtransmembrane regions of a viral protein linked to a functionalcostimulatory ligand portion. In some embodiments, the fusion proteinconsists of the portions of the viral protein which are responsible forviral entry into the cell. In some embodiments, the fusion proteinconsists of the internal domain, transmembrane region and 5-20 aminoacids of the external region of a viral protein linked to theextracellular region of a natural ligand of a costimulatory molecule.

In some embodiments, the viral protein portion is derived from alentivirus such as HIV, from a flavivirus such as yellow fever virus,hepatitis C, JEV, West Nile River Virus or hepatitis E, from a pox virussuch as avipox, fowlpox, vaccina, MVA or WR. In some embodiments, theviral protein portion is derived from influenza, rotavirus,cytomegalovirus, rabies virus. In some embodiments, the viral proteinportion is selected from the group consisting of HIV gp41, HIV gD, HIVgC, HIV gI, HCV E1, Papillomavirus L1 and Papillomavirus L2. In someembodiments, the viral protein portion is selected from the groupconsisting of flavivirus E or M protein, poxvirus E or M protein,rotavirus G protein, rabies virus G protein, influenza virus HA proteinand CMV GB protein. Importantly, the viral protein portion must containsufficient viral sequences to be assembled within the viral particlewhen the particle is assembled. Viral sequences of the fusion proteininteract with viral proteins to become included in the viral particle.

In some embodiments, the viral particle contains both a fusion proteinand a wild type envelope protein. In some embodiments, the viralparticle is free of wild type envelope protein.

In some embodiments, the fusion protein comprises two or morecostimulatory ligand portions including two costimulatory ligandportions linked by a linker 15-30 amino acids, preferably about 22 aminoacids. Such a fusion protein is particularly useful in preparingtargeted liposomes. The duplicate costimulatory ligand portions mayproceed N terminal to C terminal, linker, N terminal to C terminal whichis particularly useful since it allows for the fusion protein to beprepare by recombinant means. In some embodiments, the formula is Nterminal to C terminal, linker, C terminal to N terminal. In someembodiments, the formula is C terminal to N terminal, linker, N terminalto C terminal. In some embodiments, the formula is C terminal to Nterminal, linker, C terminal to N terminal.

In some embodiments, the fusion protein comprises one or morecostimulatory ligand portions linked to a hydrophobic tail.

In some embodiments, the fusion protein comprises one or morecostimulatory ligand portions linked to a polycationic tail, such as apolylysine tail.

In some embodiments, the fusion protein comprises a costimulatory ligandportion linked to a second portion which complexes with a protein to bedelivered. In such embodiments, the costimulatory ligand portioncomplexes to the compound directly.

Methodology and Compositions

Methods of the present invention comprise the step of administeringnon-cellular particles to tissue of the individual. In some preferredembodiments, the non-cellular particles are administeredintramuscularly, intranasally, intraperatoneally, subcutaneously,intradermally, intravenously, by aerosol administration to lung tissueor topically or by lavage to mucosal tissue selected from the groupconsisting of vaginal, rectal, urethral, buccal and sublingual.

An aspect of the present invention relates to pharmaceuticalcompositions useful in the methods of the present invention. Thepharmaceutical compositions comprise the non-cellular particles whichcomprise a compound and a costimulatory molecule or fusion protein. Thepharmaceutical compositions further comprise a pharmaceuticallyacceptable carrier or diluent. The term “pharmaceutical” is well knownand widely understood by those skilled in the art. As used herein, theterms “pharmaceutical compositions” and “injectable pharmaceuticalcompositions” are meant to have their ordinary meaning as understood bythose skilled in the art. Pharmaceutical compositions are required tomeet specific standards regarding sterility, pyrogens, particulatematter as well as isotonicity and pH. For example, injectablepharmaceuticals are sterile and pyrogen free.

In embodiments in which the pharmaceutical compositions according to thepresent invention comprise non-cellular particles which include nucleicacid molecules as the compound, a sufficient amount of non-cellularparticles are administered to introduce about 1 ng to about 10,000 μg ofnucleic acid to the tissue. In some preferred embodiments, thepharmaceutical compositions contain about 2000 μg, 3000 μg, 4000 μg or5000 μg of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 1000 μg of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 10 ng toabout 800 μg of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 0.1 to about 500 μg of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 1 to about350 μg of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 25 to about 250 μg of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 100 μg DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. Onehaving ordinary skill in the art can readily formulate a vaccine ornon-immunogenic therapeutic that comprises a genetic construct. In caseswhere intramuscular injection is the chosen mode of administration, anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation. The pharmaceutical preparations according to the presentinvention are provided sterile and pyrogen free. Pharmaceuticalcompositions according to the invention include delivery components incombination with nucleic acid molecules which further comprise apharmaceutically acceptable carriers or vehicles, such as, for example,saline. Any medium may be used which allows for successful delivery ofthe nucleic acid. One skilled in the art would readily comprehend themultitude of pharmaceutically acceptable media that may be used in thepresent invention. Suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, A. Osol, a standard reference textin this field, which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal, transdermal), oral orparenteral. Because peptides are subject to being digested whenadministered orally, oral formulations are formulated to entericallycoat the active agent or otherwise protect it from degradation in thestomach (such as prenuetralization). Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, pulmonary administration, e.g., by inhalation orinsufflation, or intrathecal or intraventricular administration. Inpreferred embodiments, parenteral administration, i.e., intravenous,subcutaneous, transdermal, intramuscular, is ordinarily used to optimizeabsorption. Intravenous administration may be accomplished with the aidof an infusion pump. The pharmaceutical compositions of the presentinvention may be formulated as an emulsion.

One skilled in the art would readily comprehend the multitude ofpharmaceutically acceptable media that may be used in the presentinvention. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield, which is incorporated herein by reference. Formulations fortopical administration may include transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Compositions forparenteral, intravenous, intrathecal or intraventricular administrationmay include sterile aqueous solutions which may also contain buffers,diluents and other suitable additives and are preferably sterile andpyrogen free. Pharmaceutical compositions which are suitable forintravenous administration according to the invention are sterile andpyrogen free. For parenteral administration, the peptides of theinvention can be, for example, formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 5% human serumalbumin. Liposomes and nonaqueous vehicles such as fixed oils may alsobe used. The vehicle or lyophilized powder may contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques. For example, a parenteralcomposition suitable for administration by injection is prepared bydissolving 1.5% by weight of active ingredient in 0.9% sodium chloridesolution

The pharmaceutical compositions according to the present invention maybe administered as a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

Dosage varies depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age,health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. Formulation of therapeuticcompositions and their subsequent administration is believed to bewithin the skill of those in the art. Usually, the dosage of peptide canbe about 1 to 3000 milligrams per 50 kilograms of body weight;preferably 10 to 1000 milligrams per 50 kilograms of body weight; morepreferably 25 to 800 milligrams per 50 kilograms of body weight.Ordinarily 8 to 800 milligrams are administered to an individual per dayin divided doses 1 to 6 times a day or in sustained release form iseffective to obtain desired results.

Preferred Components

In some embodiments, the costimulatory ligand is a fusion proteincomprising the extracellular portion of CD28 or FLT3 ligand linked to aportion of HIV gp41. The HIV gp41 portion provides for the fusionprotein to be packaged in an HIV particle, which is preferably anon-replicating particle. The CD28 extracellular portion targets theviral particle to cells that express CD80 and CD86. The FLT3 ligandportion targets the viral particle to cells that express FLT3. HIV viralparticles localized to these cells take up the viral particles. In someembodiments, the viral particles are provided with fusion proteins thatinclude Vpr sequences that provide for assembly into the viral particle.In some embodiments, the compound is a nucleic acid molecules.

In some embodiments, the compound is DNA; in some embodiments,preferably plasmid DNA. In some embodiments the compound is DNA thatcomprises a nucleotide sequences that encodes a protein operably linkedto regulatory elements functional in the cell. In some such embodimentsthe protein is an immunogenic protein, preferably in some embodiments,an immunogenic pathogen protein. In other such embodiments, thecompounds is DNA that comprises a nucleotide sequences that encodes annon-immunogenic protein operably linked to regulatory elementsfunctional in the cell. In some embodiments the compound is DNA thatcomprises a nucleotide sequences that encodes a protein operably linkedto regulatory elements derived from potent expression viral vectors suchas AAV, adenovirus or alpha viruses such as SFV. According to thisaspect, the DNA construct is provided with the potent expressionsequences within a particle that will not cross react to the immuneresponse generated against a potent expression viral vectors. This willallow boosts while taking advantage of the high expressioncharacteristics of the potent expression viral vectors. The potentexpression sequences contain the regulatory sequences, such as the ITRsequences in the case of AAV, a gene of interest and may, if theparticle is a virus, optionally include a packaging signal so that thenucleic acid molecule gets incorporated into the viral particle.

In some embodiments, the compound is a protein. In some embodiments, thecompound is a viral protein. In some embodiments, the compound is afusion protein that comprises a viral protein portion and a non-viralprotein portion.

In some embodiments, the particle is selected from the group consistingof a viral particle, a protein complex, a liposome and a cationicamphiphile/DNA complex. In some embodiments, the particle is anon-replicating viral particle.

According to some aspects of the present invention, methods ofintroducing compounds into cells are provided which comprise contactingthe cells with particles that comprises the compound and a fusionprotein. The fusion protein comprises the extracellular region of FLT-3ligand and the transmembrane and cytoplasmic regions of HIV-1 gp41. Thefusion protein provides an effective means to target the cell fordelivery of the compound.

According to some aspects of the present invention, particles comprisinga FLT-3 ligand and a therapeutic protein or nucleic acid molecule thatencodes a therapeutic protein are used to deliver therapeutic proteinsto cells. The present invention provides methods of deliveringtherapeutic proteins to an individual comprising the step ofadministering to tissue of the individual at a site on said individual'sbody, a particle that comprises therapeutic protein or a nucleic acidmolecule that encodes a therapeutic protein, and FLT-3 ligand. In someembodiments, the therapeutic protein is a non-immunogenic therapeuticprotein such as a growth factor or cytokine. In some embodiments, thetherapeutic protein is a fusion protein that comprise a viral portionthat facilitates packaging the fusion protein in the particle and annon-viral portion such as a transcription factor, growth factor,chemokine or cytokine portion. The protein or DNA encoding the proteinare provided as part of/within the particle. In some embodiments, DNAprovided as part of/within the particle is plasmid DNA. In someembodiments, the particle is selected from the group consisting of aviral particle, a protein complex, a liposome and a cationicamphiphile/DNA complex. In some embodiments, the particle is anon-replicating viral particle.

Some embodiments of the invention provide methods of immunizing againstcancer comprising administering to an individual, a cancer cellcomprising a recombinant expression vector that encodes a FLT-3 ligand.Some embodiments of the invention relate to cancer cells that comprisinga recombinant expression vector that encodes a FLT-3 ligand.

According to some embodiments of the invention, a particle thatcomprises a compound and a FLT-3 ligand is provided. In someembodiments, the FLT-3 ligand is a fusion protein comprising theextracellular region of the natural FLT-3 ligand and the transmembraneand cytoplasmic regions of retrovirus envelope protein. In someembodiments, the FLT-3 ligand is a fusion protein comprising theextracellular region of the natural FLT-3 ligand and the transmembraneand cytoplasmic regions of HIV-1 gp41. In some embodiments, the FLT-3ligand is a fusion protein comprising the extracellular region of thenatural FLT-3 ligand and the transmembrane and cytoplasmic regions ofHerpes virus gD, gB, gH or gL. In some embodiments, the compound is anucleic acid or protein. In some embodiments, the compound is DNA. Insome embodiments, the compound is plasmid DNA. In some embodiments, thecompound is DNA that comprises a nucleotide sequences that encodes aprotein operably linked to regulatory elements functional in the cell.In some embodiments, the compound is DNA that comprises a nucleotidesequences that encodes an immunogenic protein operably linked toregulatory elements functional in the cell. In some embodiments, thecompound is DNA that comprises a nucleotide sequences that encodes animmunogenic pathogen protein operably linked to regulatory elementsfunctional in the cell. In some embodiments, the compound is DNA thatcomprises a nucleotide sequences that encodes an non-immunogenic proteinoperably linked to regulatory elements functional in the cell. In someembodiments, the compound is a protein. In some embodiment, the compoundis a fusion protein that comprises a viral portion that facilitatespackaging the fusion protein in the particle and an non-viral portionsuch as a transcription factor, growth factor, chemokine or cytokineportion. In some embodiments, the viral protein portion is the portionof HIV Vpr protein that binds to Gag resulting in Vpr protein beingincluded in the HIV particle. According to such embodiments, the Vprprotein portion facilitates the inclusion of the fusion protein in theparticle. In some embodiments, the particle is selected from the groupconsisting of a viral particle, a protein complex, a liposome and acationic amphiphile/DNA complex. In some embodiments, the particle is anon-replicating viral particle. In some embodiments, the particle is anon-replicating HIV or Herpes viral particle.

Transcription factors or portions thereof included as therapeuticproteins or as part of fusion proteins drive and/or modulate geneexpression in cells infected by the particles. T-bet is an example oftranscription factors which shuts down Th2 responses and promotes Th1responses. Such an example would be useful in a vaccine. Gata is anexample of transcription factors which shut down Th1 responses andpromotes Th2 responses. Such an example would be useful in a treatmentof autoimmune disease.

Cytokines or portions thereof included as therapeutic proteins or aspart of fusion proteins, drive/modulate immune responses as dochemokines or portions thereof included as therapeutic proteins or aspart of fusion proteins. An example of a cytokine is IL-15. An exampleof a chemokine is RANTES.

Therapeutic and fusion proteins may include transport proteins orfragments thereof and processing proteins of fragments thereof. Anexample of a transport protein is p70. An example of a processingprotein is Tap.

In the case of HIV derived particles which packages Vpr protein andfusion proteins which contain Vpr portions, the particles can contain asmany as 2400 copies of the fusion protein. The Vpr portion is inactive,it does not contain sequences which induce cell cycle arrest andapoptosis.

The fusion protein may include a protease cleavage site between the twoportions. An example of such a cleavage site s the cleavage siterecognized by the HIV protease. In addition to HIV-derived particles,the invention is envisioned to include particles and chimerics basedupon other lentiviruses and lentivirus proteins. The invention alsopertains to the use of free vpr fusions to be delivered as drugs aswell.

A further aspect of the invention relates to methods of immunizingindividuals. Such comprise the steps of administering to tissue of theindividual at a site on the individual's body, a DNA molecule thatcomprises a nucleotide sequence that encodes an immunogenic proteinoperably linked to regulatory elements. Subsequently, a particle thatcomprises an immunogenic protein is administered to the individual. Insome embodiments, the particle may further comprises a compound. In someembodiments, the compound maybe a nucleic acid molecule. In someembodiments, the compound is DNA. In some embodiments, the compound isplasmid DNA. In some embodiments, the compound is DNA that comprises anucleotide sequences that encodes an immunogenic protein operably linkedto regulatory elements functional in the cell. In some embodiments, thecompound is DNA that comprises a nucleotide sequences that encodes animmunogenic pathogen protein operably linked to regulatory elementsfunctional in the cell. In some embodiments, the compound is DNA thatcomprises a nucleotide sequences that encodes an non-immunogenic proteinoperably linked to regulatory elements functional in the cell. In someembodiments, the particle is a viral particle. In some embodiments, theparticle is a non-replicating viral particle. In some embodiments, theparticle is a protein complex.

According to some embodiments, the compounds is a protein which includesviral sequences which function to package the compound in the viralparticle. In some embodiments, the viral sequences are viral proteins.In some embodiments, the viral sequences are fragments of viral proteinswhich retain their ability to complex with other viral proteins in theassembly of viral particles. In some embodiments, the particle is an HIVparticle and the compound is a fusion protein which includes sequencesof the HIV Vpr protein. The fusion protein which includes sequences ofthe HIV Vpr protein are packaged in the HIV particle. In someembodiment, the compound is a fusion protein that comprises a viralportion that facilitates packaging the fusion protein in the particleand an non-viral portion such as a transcription factor, growth factor,chemokine or cytokine portion. In some embodiments, the viral proteinportion is the portion of HIV Vpr protein that binds to Gag resulting inVpr protein being included in the HIV particle. According to suchembodiments, the Vpr protein portion facilitates the inclusion of thefusion protein in the particle.

Example 3

The present invention relates to compositions useful for deliveringfusion proteins into specifically targeted cells. The fusion proteincomprises a biologically active portion and a Vpr fragment which bindsto HIV viral proteins assembled as part of the viral particle. In someembodiments, the particle additionally contains cell-type specific coatprotein to deliver the particle specific to cells that the coat proteinbinds to. The present invention relates to the fusion proteins, to theparticles, to the pharmaceutical compositions that comprise theparticles and pharmaceutically acceptable carriers, to the nucleic acidmolecules that encode the components, to the expression vectors and hostcells that contain the nucleic acid molecules and to the methods ofproducing and using the compositions.

To prepare a fusion drug delivery particle of the invention, theenvelope protein (Env) of a retrovirus is chosen based upon the celltype such a retrovirus infects. Cell specific envelope proteins are wellknown. A chimeric gene is designed which includes the portion of the Vprprotein that is required for Vpr to be incorporated into the viralparticle together with a biological active protein which retains itsactivity when linked to the portion of vpr. Cells are co-transfectedwith a nucleic acid molecule that encodes the desired env, the chimericgene that encodes the fusion protein, a nucleic acid molecule thatencodes p24 or a nucleic acid molecule that encodes the full length gagprecursor plus the HIV protease. Expression of these sequences willresult in the proteins thus encoded being produced and assembly of thedrag delivery particle. Noncoding RNA may also be provided for safetysince the assembling particle will package RNA.

The particles may be those such as described in Example 2 which comprisethe Vpr fusion protein, particles essentially as described in Example 2which comprise the Vpr fusion protein but which do not contain fusionproteins which have co-stimulatory molecule ligands, particles which areproduced by packaging cell lines that produce HIV-derived particles asdescribed in the packaging systems incorporated above which includingthe chimeric gene that encodes the Vpr fusion protein, particles whichare produced by packaging cell lines that contain heterologous Envproteins or fusion Env proteins as described in U.S. Pat. No. 5,714,316.

Biologically active proteins which can be used in fusion proteinsinclude transcription factors, growth factors, cytokines, chemokines,transport proteins and processing proteins.

Example 4 Recombinant Cell Based Cancer Vaccines

Another aspect of the present invention relates to the use ofrecombinant cancer cells as cancer vaccines. The use of recombinantcancer cells as cancer vaccines is described in U.S. Pat. No. 5,935,569,which is incorporated by references. According to this aspect of theinvention, the recombinant gene expressed by the cancer cell is a Flt-3ligand. The cancer vaccine is an autologous cancer cell transfected withan expression vector that comprises a sequence encoding a Flt-3 ligand.The cancer cells expressing the Flt-3 ligand are targeted to cells thatexpress Flt-3 molecules and the immune response against the cancer cellsin enhanced. In preferred embodiments, the recombinant expression vectorthat comprises a sequence encoding a Flt-3 ligand is transfected intocancer cells ex vivo and the transfected cells are then restored to thepatient.

In some embodiments, the transfected cancer cell is further providedwith an expression vector that includes a nucleotide sequence thatencodes a death domain receptor or death domain signal or a toxin. Deathdomain receptors include, but are not limited to; Apo-1 (Oehm et al., J.Biol. Chem., 1992, 267(15), 107Q9-15; Accession Number X63717); Fas(Itoh et al., Cell, 1991, 66(2),233-43; Accession Number M67454); TNFR-1(Nophar et al., EMBO J., 1990, 9(10), 3269-78; Accession Number M67454);p55 (Loetscher et al., Cell, 1990, 61, 351-359; Accession NumbersM58286, M33480); WSL-1 (Kitson et al., Nature, 1996, 384(6607), 372-5;Accession Number Y09392); DR3 (Chinnaiyan et al., Science, 1996, 274(5829), 990-2; Accession Number U72763); TRAMP (Bodmer et al., Immunity,1997, 6(1), 79-88; Accession Number U75381); Apo-3 (Marsters et al.,Curr. Biol., 1996, 6(12), 1669-76; Accession Number U7461 1); AIR(Degli-Esposti et al., direct submission, Accession Number U78029); LARD(Screaton et al., Proc. Natl. Acad. Sci. USA, 1997, 94(9), 4615-19;Accession Number U94512); NGRF (Johnson et al., Cell, 1986,47(4),545-554; Accession Number M14764); DR4 (Pan et al., Science, 1997,276(5309), 111-113; Accession Number U90875); DR5 (Sheridan et al.,Science, 1997, 277(5327), 818-821; Accession Number AF012535); KILLER(Wu et al., Nature Genetics, in press,; TRAIL-R2 (MacFarlane et al, J.Biol. Chem., 1997, in press; Accession Number AF020501); TRICK2(Screaton et al., Curr. Biol., 1997, in press; Accession NumberAF018657); DR6 (Pan et al., unpublished; Accession Number AF068868).Death signals, i.e. proteins that interact with the death domainreceptors include, but are not limited to; FADD (Chinnaiyan et al.,Cell, 1995, 81(4), 505-12; Accession Number U24231); FAP-1 (Sato et al.,Science, 1995, 268 (5209), 411-15; Accession Number L34583); TRADD (Hsuet al., Cell, 1995, 81(4), 495-504; Accession Number L41690); RIP(Stanger et al., Cell, 1995, 81(4), 513-23; Accession Number U25994);and FLICE (Muzio et al., Cell, 1996, 85(6); 817-27; Accession NumberU58143); RAIDD (Lennon et al., Genomics, 1996, 33(1), 151-2; AccessionNumber U79115). Death signals also include ligands that bind deathdomain receptors and initiate apoptosis include, but are not limited to;FAS-L (Alderson et al., J. Exp. Med., 1995, 181(1),71-7; AccessionNumber U08137), and TNF, and mediators that interact with death domainreceptors include, but are not limited to; FADD (Chinnaiyan et al.,Cell, 1995, 81(4),505-12; Accession Number U24231); MORT1 (Boldin etal., J. Biol. Chem., 1995, 270(14),7795-8; Accession Number X84709);CRADD (Ahmad et al., Cancer Res., 1997, 57(4), 615-9; Accession NumberU84388); and MyD88 (Bonnert et al, FEBS Lett., 1997, 402(1), 81-4;Accession Number U84408). Toxins include proteins which kill cells.Toxins include but are not limited to insect and snake venoms, bacterialendotoxins such as Psuedomoneus endotoxin, double chain ribosomeinactivating proteins such as ricin including single chain toxin, andgelonin. TABLE 1 Picornavirus Family Genera: Rhinoviruses: (Medical)responsible for ˜50% cases of the common cold. Enteroviruses: (Medical)includes polioviruses, Coxsackieviruses, echoviruses, and humanenteroviruses such as hepatitis A virus. Apthoviruses: (Veterinary)these are the foot and mouth disease viruses. Target antigens: VP1, VP2,VP3, VP4, VPG Calcivirus Family Genera: Norwalk Group of Viruses:(Medical) these viruses are an important causative agent of epidemicgastroenteritis. Togavirus Family Genera: Alphaviruses: (Medical andVeterinary) examples include Sindbis viruses, RossRiver virus andEastern & Western Equine encephalitis. Rubivirus: (Medical) Rubellavirus. Flariviridue Family Examples include: (Medical) dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Hepatitis C Virus: (Medical) these viruses are notplaced in a family yet but are believed to be either a togavirus or aflavivirus. Most similarity is with togavirus family. CoronavirusFamily: (Medical and Veterinary) Infectious bronchitis virus (poultry)Porcine transmissible gastroenteric virus (pig) Porcine hemagglutinatingencephalomyelitis virus (pig) Feline infectious peritonitis virus (cats)Feline enteric coronavirus (cat) Canine coronavirus (dog) The humanrespiratory coronaviruses cause ˜40 cases of common cold. EX. 224E, 0C43Note - coronaviruses may cause non-A, B or C hepatitis Target antigens:E1 - also called M or matrix protein E2 - also called S or Spike proteinE3 - also called HE or hemagglutin-elterose glycoprotein (not present inall coronaviruses) N - nucleocapsid Rhabdovirus Family Genera:Vesiculovirus: Vesicular Stomatitis Virus Lyssavirus: (medical andveterinary) rabies Target antigens: G protein N protein FiloviridueFamily: (Medical) Hemorrhagic fever viruses such as Marburg and Ebolavirus Paramyxovirus Family: Genera: Parainfluenza Virus Type 1Parainfluenza Virus Type 3 Bovine Parainfluenza Virus Type 3Rubulavirus: (Medical and Veterinary) Mumps virus, Parainfluenza VirusType 2, Parainfluenza Virus Type 4, NewCastle disease virus (importantpathogen in chickens) Morbillivirus: (Medical and Veterinary) Measles,canine distemper Pneumonvirus: (Medical and Veterinary) Respiratorysyncytial virus Orthomyxovirus Family (Medical) The Influenza virusBunyavirus Family Genera: Bunyavirus: (Medical) California encephalitis,La Crosse Phlebovirus: (Medical) Rift Valley Fever Hantavirus: Puremalais a hemahagin fever virus Nairovirus (Veterinary) Nairobi sheep diseaseAlso many unassigned bungaviruses Arenavirus Family (Medical) LCM, Lassafever virus Reovirus Family Genera: Reovirus: a possible human pathogenRotavirus: acute gastroenteritis in children Orbiviruses: (Medical andVeterinary) Cultivirus: Colorado Tick fever, Lebombo (humans) equineencephalosis, blue tongue Retrovirus Family Sub-Family: Oncorivirinal:(Veterinary) (Medical) feline leukemia virus, HTLVI and HTLVIILentivirinal: (Medical and Veterinary) HIV, feline immunodeficiencyvirus, equine infections, anemia virus Spumavirinal Papovavirus FamilySub-Family: Polyomaviruses: (Medical) BKU and JCU viruses Sub-Family:Papillomavirus: (Medical) many viral types associated with cancers ormalignant progression of papilloma Adenovirus (Medical) EX AD7, ARD.,O.B. - cause respiratory disease - some adenoviruses such as 275 causeenteritis Parvovirus Family (Veterinary) Feline parvovirus: causesfeline enteritis Feline panleucopeniavirus Canine parvovirus Porcineparvovirus Herpesvirus Family Sub-Family: alphaherpesviridue Genera:Simplexvirus (Medical) HSVI, HSVII Varicellovirus: (Medical -Veterinary) pseudorabies - varicella zoster Sub-Family -betaherpesviridue Genera: Cytomegalovirus (Medical) HCMV MuromegalovirusSub-Family: Gammaherpesviridue Genera: Lymphocryptovirus (Medical) EBV -(Burkitts lympho) Rhadinovirus Poxvirus Family Sub-Family:Chordopoxviridue (Medical - Veterinary) Genera: Orthopoxvirus Variola(Smallpox) Vaccinia (Cowpox) Parapoxivirus - Veterinary Auipoxvirus -Veterinary Capripoxvirus Leporipoxvirus Suipoxvirus Sub-Family:Entemopoxviridue Hepadnavirus Hepatitis B virus Family: Unclassified:Hepatitis delta virus

TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci include:pneumococcal; staphylococcal; and streptococcal. Pathogenicgram-negative cocci include: meningococcal; and gonococcal. Pathogenicenteric gram-negative bacilli include: enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigellosis;hemophilus; moraxella; chancroid; brucellosis; tularemia; yersinia(pasteurella); streptobacillus moniliformis and spirillum; listeriamonocytogenes; erysipelothrix rhusiopathiae; diphtheria; cholera;anthrax; donovanosis (granuloma inguinale); and bartonellosis.Pathogenic anaerobic bacteria include: tetanus; botulism; otherclostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenicspirochetal diseases include: syphilis; treponematoses: yaws, pinta andendemic syphilis; and leptospirosis. Other infections caused by higherpathogen bacteria and pathogenic fungi include: actinomycosis;nocardiosis; cryptococcosis, blastomycosis, histoplasmosis andcoccidioidomycosis; candidiasis, aspergillosis, and mucormycosis;sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis,mycetoma and chromomycosis; and dermatophytosis. Rickettsial infectionsinclude rickettsial and rickettsioses. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes Pathogenic protozoans and helminths and infections therebyinclude: amebiasis; malaria; leishmaniasis; trypanosomiasis;toxoplasmosis; pneumocystis carinii; babesiosis; giardiasis;trichinosis; filariasis; schistosomiasis; nematodes; trematodes orflukes; and cestode (tapeworm) infections.

1. A method of introducing a compound into a cell that expresses Flt3molecules, said method comprising contacting the cell with anon-cellular particle that comprises the compound and a Flt-3 ligand. 2.The method of claim 1 wherein the compound is a nucleic acid molecule orprotein.
 3. The method of claim 1 wherein the compound is DNA.
 4. Themethod of claim 1 wherein the compound is DNA that comprises anucleotide sequences that encodes a protein operably linked toregulatory, elements functional in the cell.
 5. The method of claim 1wherein the compound is DNA that comprises a nucleotide sequences thatencodes an immunogenic protein operably linked to regulatory elementsfunctional in the cell.
 6. The method of claim 1 wherein the compound isDNA that comprises a nucleotide sequences that encodes annon-immunogenic protein operably linked to regulatory elementsfunctional in the cell.
 7. The method of claim 1 wherein the compound isDNA that comprises a nucleotide sequences that encodes a proteinoperably linked to regulatory elements derived from AAV, adenovirus oralphavirus.
 8. The method of claim 1 wherein the compound is DNA thatcomprises a nucleotide sequences that encodes a protein operably linkedto regulatory elements derived from AAV, adenovirus or alphavirus, saidDNA further comprising a packaging signal to facilitate itsincorporation into said particle.
 9. The method of claim 1 wherein thecompound is a viral protein.
 10. The method of claim 1 wherein thecompound is a fusion protein comprising a an HIV Vpr portion whichfacilitate incorporation of said fusion protein into said particle. 11.The method of claim 10 wherein fusion protein comprising a proteasecleavage site between said Vpr portion and a biologically activeportion.
 12. The method of claim 11 wherein the protease cleavage siteis a cleavage site recognized by HIV-1 protease.
 13. The method of claim10 wherein fusion protein comprising a biologically active portionselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins.
 14. The method of claim 13 wherein fusion protein comprising abiologically active portion selected from the group consisting oftranscription factor Tbet, transcription factor Tgata, cytokine IL-15,chemokine Rantes, transport protein p70 and processing protein TAP. 15.The method of claim 1 wherein the particle is a viral particle, aprotein complex, a liposome or a cationic amphiphile/DNA complex. 16.The method of claim 1 wherein the particle comprises a fusion proteincomprising Flt-3 ligand or a FLT-3 binding fragment thereof and thetransmembrane and cytoplasmic regions of HIV-1 gp41.
 17. A non-cellularparticle that comprises a fusion protein comprising Flt-3 ligand or aFLT-3 binding fragment thereof.
 18. The particle of claim 17 furthercomprising a nucleic acid molecule and/or fusion protein.
 19. Theparticle of claim 18 comprising DNA.
 20. The particle of claim 19wherein the DNA comprises a nucleotide sequences that encodes a proteinoperably linked to regulatory elements functional in the cell.
 21. Theparticle of claim 19 wherein the DNA comprises a nucleotide sequencesthat encodes an immunogenic protein operably linked to regulatoryelements functional in the cell.
 22. The particle of claim 19 whereinthe DNA comprises a nucleotide sequences that encodes an non-immunogenicprotein operably linked to regulatory elements functional in the cell.23. The particle of claim 19 wherein the DNA comprises a nucleotidesequences that encodes a protein operably linked to regulatory elementsderived from AAV, adenovirus or alphavirus.
 24. The particle of claim 19wherein the DNA comprises a nucleotide sequences that encodes a proteinoperably linked to regulatory elements derived from AAV, adenovirus oralphavirus, said DNA further comprising a packaging signal to facilitateits incorporation into said particle.
 25. The particle of claim 18comprising a fusion protein.
 26. The particle of claim 18 wherein thefusion protein comprises an HIV Vpr portion which facilitateincorporation of said fusion protein into said particle.
 27. Theparticle of claim 18 wherein the fusion protein comprises a proteasecleavage site between said Vpr portion and a biologically activeportion.
 28. The particle of claim 27 wherein the protease cleavage siteis a cleavage site recognized by HIV-1 protease.
 29. The particle ofclaim 18 wherein fusion protein comprising a biologically active portionselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins.
 30. The particle of claim 29 wherein fusion protein comprisinga biologically active portion selected from the group consisting oftranscription factor Tbet, transcription factor Tgata, cytokine IL-15,chemokine Rantes, transport protein p70 and processing protein TAP. 31.The particle of claim 17 wherein the particle is a viral particle, aprotein complex, a liposome or a cationic amphiphile/DNA complex. 32.The particle of claim 17 wherein the particle comprises a fusion proteincomprising Flt-3 ligand or a FLT-3 binding fragment thereof and thetransmembrane and cytoplasmic regions of HIV-1 gp41.
 33. The method ofimmunizing an individual comprising administering a particle of claim30.
 34. The method of treating an individual for autoimmune diseasecomprising administering a particle of claim
 30. 35. A non-cellularparticle that comprises a fusion protein comprising a Vpr portion and abiologically active non-Vpr portion.
 36. The particle of claim 35wherein the fusion protein comprises an HIV Vpr portion which facilitateincorporation of said fusion protein into said particle.
 37. Theparticle of claim 35 wherein the fusion protein comprises a proteasecleavage site between said Vpr portion and a biologically activeportion.
 38. The particle of claim 37 wherein the protease cleavage siteis a cleavage site recognized by HIV-1 protease.
 40. The particle ofclaim 35 wherein fusion protein comprising a biologically active portionselected from the group consisting of transcription factors, growthfactors, cytokines, chemokines, transport proteins and processingproteins.
 41. The particle of claim 40 wherein fusion protein comprisinga biologically active portion selected from the group consisting oftranscription factor Tbet, transcription factor Tgata, cytokine IL-15,chemokine Rantes, transport protein p70 and processing protein TAP. 42.The particle of claim 35 further comprising a nucleic acid moleculeand/or fission protein.
 43. The particle of claim 42 comprising DNA. 44.The particle of claim 43 wherein the DNA comprises a nucleotidesequences that encodes a protein operably linked to regulatory elementsfunctional in the cell.
 45. The particle of claim 43 wherein the DNAcomprises a nucleotide sequences that encodes an immunogenic proteinoperably linked to regulatory elements functional in the cell.
 46. Theparticle of claim 43 wherein the DNA comprises a nucleotide sequencesthat encodes an non-immunogenic protein operably linked to regulatoryelements functional in the cell.
 47. The particle of claim 43 whereinthe DNA comprises a nucleotide sequences that encodes a protein operablylinked to regulatory elements derived from AAV, adenovirus oralphavirus.
 48. The particle of claim 43 wherein the DNA comprises anucleotide sequences that encodes a protein operably linked toregulatory elements derived from AAV, adenovirus or alphavirus, said DNAfurther comprising a packaging signal to facilitate its incorporationinto said particle.
 49. The particle of claim 35 comprising acostimulatory ligand or a fusion protein comprising costimulatory ligandportion.
 50. The particle of claim 35 wherein the particle is an HIVviral particle.
 51. The particle of claim 35 wherein the particle is anHIV viral particle with a modified or heterologous Env protein.
 52. Themethod of delivering a fusion protein to an individual administering aparticle of claim
 35. 53. A non-cellular particle that comprises anucleotide sequences that encodes a protein operably linked toregulatory elements derived from AAV, adenovirus or alphavirus whereinwhen said particle is a virally derived particle, said regulatoryelements are derived from a different virus than said particle.
 54. Theparticle of claim 53 wherein said particle is derived particle from alentivirus.
 55. The particle of claim 53 wherein said nucleotidesequences further comprises a packaging signal which facilitatespackaging of nucleic acid molecules by said particle.
 56. A method ofdelivering and expressing DNA to cells of an individual that comprisesadministering to said individual a particle of claim
 53. 57. Anucleotide sequences that encodes a protein operably linked toregulatory elements derived from AAV, adenovirus or alphavirus and apackaging signal which facilitates packaging of nucleic acid moleculesby viral particles different from the viral particles from which theregulatory sequences have been derived.